This invention relates to a sensitive method of determining the number of cells in cell culture, that is, a method which is adaptable to determining cell counts in levels as low as about 100 to about 1000 cells.
Various process parameters are of substantial importance in cell and tissue culture. One of these parameters is the determination of the number of cells or cell count. This is an important quantitative criterion for estimating the amount of growth or survival in a cell population during culture or maintenance of the cells.
Cells can be determined directly by enumeration of the cell number by means of a haemocytometer, a microscope or an electronic particle counter such as, for example, a Coulter.RTM. electronic cell counter.
One method of estimating viable cells is to determine the proportion of individual cells which can give rise to cell colonies, that is, by determining the plating efficiency. However, this generally is a slow and cumbersome process.
Indirect determination of cells can be made by measurement of packed cell volume, by chemical determination of a cellular component, for example, protein or deoxyribonucleic acid, or by uptake of a chromogenic dye such as neutral red. Another method of determination involves measurement of cellular lactate dehydrogenase activity by reaction with the coenzyme AND, which can be followed spectrophotometrically, as described, for example, by Jauregui et al., In Vitro 17(12), 1100-1110 (1981).
Although the foregoing methods are useful in many cases, they have been found inconvenient or unsuitable in certain instances either because they are time consuming, cumbersome or not adaptable to the determination of large numbers of samples, especially where the cells are grown under very sparse conditions and where sensitivity is an important consideration. That is, conventional methods are not generally adaptable to determining the number of cells in cell culture at sensitivity as low as about 100 to about 1000 cells, but, instead, are usually adaptable only to higher cell counts.